Telomeres are special DNA-protein complexes at the end of chromosomes, whose major function is to protect chromosomes from degradation and end-to-end fusion, therefore, are essential for genome stability and cell survival (Blackburn, E. H., 2000, Nat. Struct. Biol., 7: 847-850; Counter et al., 1992, EMBO J., 11: 1921-1929). Acquiring the ability for telomere stabilization through telomerase reactivation has been proposed to be responsible for the immortal malignant phenotype of most cancer cells (Shay et al., 2001, Human Molecular Genetics, 10: 677-685).
Telomerase plays a pivotal role for the complete replication of the terminal telomeric DNA, which is a long stretch repetitive DNA sequence (TTAGGG in mammals). Telomerase activity is absent in most somatic cells in adult human tissues. Telomere lengths in normal cells shorten with each cell division, and progressive telomere shortening eventually results in cell senescence or crisis due to telomere dysfunction, characterized by mass genome instability (e.g. widespread chromosome end-to-end fusions), senescence-like growth arrest and apoptosis (Blackburn, E. H., 2000, Nature (Lond.), 408: 53-56; Blackburn, E. H., 2001, Cell, 106: 661-673). In more than 85% of all human cancer cells, telomerase is reactivated (Shay et al., 1997, J. Cancer, 33: 787-791), and the activation of telomerase activity has been shown to prevent telomere shortening, thereby immortalize cells (Bordnar et al., 1998, Science (Wash. D.C.), 297: 349-352; Vaziri et al., 1998, Curr. Biol., 8: 279-282). The expression of telomerase has also been demonstrated to be required both for the malignant transformation of normal human cells (Elenbaas et al., 2001, Genes Dev., 15: 50-65; Hahn et al., 1999, Nature (Lond.), 499: 464-468; Rich et al., 2001, Cancer Res., 61: 3556-3560) and the continued proliferation of cancer cells (Hahn et al., 1999, Nat. Med., 5: 1164-1170, Wu et al., 1999, Nat. Genet., 21: 220-224).
Telomerase is a ribonucleoprotein (RNA) complex with the telomerase RNA component (TR) and the catalytic protein subunit the telomerase reverse transcriptase (TERT) as its two core elements, which are conserved among all species (Harrington et al., 1997, Genes Dev., 11: 3109-3115; Nakamura et al., 1997, Science (Wash. DC), 277: 955-959; Weinrich et al., 1997, Nat. genet., 17: 498-505). The well-documented biological activity of TERT is to reverse transcribe the TR template into telomeric DNA sequence and elongate the telomere length. Therefore, TERT is a special reverse transcriptase. The TR gene is ubiquitously expressed in all human tissue (Avilion et al., 1996, Cancer Res., 56: 645-650, Feng et al., 1995, Science (Wash. DC), 269: 1236-1241), while the hTERT gene expression is restricted to only telomerase positive cells (e.g. germ lines, stem cells and most cancer cells) (Kilian et al, 1997, Hum. Mol. Genet., 6: 2011-2019), indicating that the expression of hTERT is a rate-limiting step for human cellular telomerase activity (Meyerson et al., 1997, Cell, 90: 785-795). Indeed, illegitimately induced expression of the TERT gene has been found to be the cause for telomerase reactivation in cancer cells. Consistently, many oncogenic proteins (such as c-Myc) act as the transcriptional activators for the TERT gene (Greenberg et al., 1999, Oncogene, 18: 1219-1226; Wu et al., 1999, Nat. Genet., 21: 220-224).
The functional domains of the hTERT protein include the hTR specific binding domain and the conserved reverse transcriptase motifs (RT-motifs), which are located at its N-terminal and the central regions of the protein, respectively (Bachand et al., 2001, Mol. Cell. Biol., 21: 1888-1897; Lai et al., 2001, Mol. Cell. Biol., 21: 990-1000; Nakamura et al., 1998, Cell, 92: 587-590). Both domains are required for the enzymatic activity of all TERT. Besides its hTR association and its reverse transcription activity, hTERT also needs to be localized to the nucleus to be functional. In yeast cells, this process is regulated by the specific interactions between the yeast telomerase-associated protein Est1 and the yeast telomeric single strand DNA specific binding protein Cdc13 (Evans et al., 1999, Science (Wash. DC), 286: 117-120, Lustig et al, 2001, Nat. Struct. Biol., 8: 297-299). The mechanism for recruiting hTERT to telomere end in human cells, however, has not been elucidated, neither is the human counter part for Est1 or Cdc13.
Because telomere stabilization through hTERT activation is critical for the long-term survival of cancer cells, specific interruption of this event may represent an excellent anticancer strategy. Up to date, most efforts were focused on inhibiting telomerase activity, thus leading to telomere shortening and ultimately inducing telomere dysfunction in cancer cells. The most commonly used strategies are either to specifically destroy the RNA template (TR) (Herbert et al., 1998, Proc. Natl. Acad. Sci. USA, 96:14276-14281; Kondo et al., 1998, Oncogene, 16: 3323-3330, Yokoyama et al., 1998, Cancer Res., 58: 5406-5410) or to inhibit the catalytic activity of TERT (Hahn et al., 1999, Nat. Med., 5: 1164-1170). These efforts have effectively led to telomerase suppression, progressive telomere shortening, telomere dysfunction, and ultimately marked inhibition of cell growth both in vitro and in vivo. Nevertheless, the inhibition of telomerase activity cause cancer cells to undergo a process to elicit the alternative lengthening of telomere (ALT) mechanism (Bryan et al., 1995, EMBO J. 14: 4240-4248), which is a telomerase-independent telomere maintaining pathway normally suppressed by telomerase activity (Ford et al., 2001, J Biol. Chem., 276: 32198-32203; Grobelny et al., 2001, Hum. Mol. Genet. 10: 1953-1961). This ALT mechanism uses the recombination-based pathway for telomere maintenance, and the activation of this pathway has previously been demonstrated in human cancer cells that are telomerase negative (Bryan et al., 1997, Nat. Med., 3: 1271-1274; Dunham et al., 2000, Nat. Genet., 26: 447-450).
Thus, there is a need for a better strategy that is more effective and less prone to the development of resistance. The present invention provides an approach that can produce a more favorable clinical outcome.